The Immune System
The major function of the immune system is to protect the host of invading pathogens. A number of different cell types, both antigen-independent and antigen-specific, have evolved to detect and neutralize these invading pathogens. Among them, lymphocytes have an important characteristic, which is their ability to specifically recognize antigens, a feature not possessed by any other cell. This means that any lymphocyte function stimulated by an antigen is directed solely at that antigen.
Lymphocytes may be divided into two major populations: T and B. T lymphocytes have a central role in regulating the immune response and for this they produce and secrete lymphokines (i.e.: interleukins). B-lymphocytes are the only cells that produce antibodies, which are proteins—Immunoglobulins (IgG)—that recognize and bind antigens.
Some T lymphocytes are known as helper (Th lymphocytes) because they assist B cells to produce antibody. T-lymphocytes express a characteristic membrane molecule designated CD4. Other T lymphocytes are known as cytotoxic (CTL) because they are capable of killing certain cells. They express a different characteristic membrane protein designated CD8.
Th lymphocytes, in mice, have been subdivided according to the lymphokines they produce in groups designated Th0, Th1 and Th2. In general, Th 1 lymphocytes produce lymphokines which stimulate macrophages and CTLS (IL2, IFNγ, TNF-β), Th 2 lymphocytes produce lymphokines which stimulate B lymphocytes to proliferate and produce antibody (IL 2, IL5, IL6, IL10, IL13), whilst Th 0 lymphocytes produce a mixture of lymphokines and are thought to be an intermediate stage from which Th 1 and Th 2 lymphocytes are derived. In humans, Th1 and Th2 like lymphocytes have been demonstrated, although they do seem to show a less strict division with respect to their patterns of cytokine secretion.
A third population of lymphocytes, which lack the major makers of T and B cells include the natural killer cells (NK cells), the killer cells (K cells) and the lymphokine-activated killer cells (L A K cells). NK cells can kill certain tumor cells and some virally infected cells, but unlike cytotoxic T lymphocytes they are not capable of recognizing a specific antigen. K cells are able to bind to cells, which have antibody to them via their antigen-binding regions and kill them. L A K cells do not specifically recognize an antigen but they are capable of destroying a wider range of targets a NK cells.
Macrophages and dendritic cells play a critical role in initiating immune responses, helping T cells to respond to antigens.
There are several antibody classes. The IgG class comprises most of the circulating antibodies and it has four subclasses designated IgG1, IgG2, IgG3 and IgG4.
The IgM class comprises about 10% of the circulating antibodies. These are the principal antibodies produce during the primary immunological response. The IgA class comprises most of the antibody secreted at mucous membranes and exerts its protective effect by blocking access of the antigen to the inner body. The IgD class comprises less than 1% of serum antibodies and its biological role is largely unknown. The IgE class comprises antibodies that are mainly bound to the surface of most cells and basophils. These antibodies are associated with reactions that occur in individuals who are undergoing allergic reactions.
Vaccines and Vaccines Adjuvants
Vaccines are preparations used to stimulate animals to mount an immune response against antigens included in the vaccine.
Vaccines often include adjuvants, which are substances that used in combination with specific antigen produce more immunity than the antigen used alone. (Ramon, G., 1926. Procedes pour accroite Ia production des antitoxins. Ann. Inst. Pasteur. 40, 1-10).
Many kind of compounds function as vaccine adjuvants (Edelman, R., 2000. An overview of adjuvant use, in: Vaccine Adjuvants. Preparation Methods and Research Protocols. D. T. O'Hagan, Ed., Humana Press, Totowa, N.J. References cited in this article are incorporated herein as background material).
However, currently, the only adjuvants approved for use in humans are aluminum salts (Gupta, R. K. and Rost, B. E., 2000. Aluminum compounds as vaccine adjuvants in: Vaccine Adjuvants. Preparation Methods and Research Protocols. D. T. O'Hagan, Ed., Humana Press, Totowa, N.J.) and the oil-in-water emulsion M F 59 (Ott, G. Radhakrishman, R. Fang, J. and Hora, M., 2000. The adjuvant M F 59: A 10-Year Perspective, in : Vaccine Adjuvants. Preparation Methods and Research Protocols. D. T. O'Hagan, Ed., Humana Press, Totowa, N.J.).
Nucleic Acids as Immunostimulatory Compounds
Several polynucleotides have been demonstrated to have immunostimulatory properties. For example, poly (I,C) is an inducer of interferon (IFN) production, macrophage activation and NK cell activation (Talmadge, J. E., Adams, J., Phillips, H., Collins, M., Lenz, B., Schneider, M., Schlick, E., Ruffmann, R., Wiltrout, R. H., Chirigos, M. A. 1985. Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L:-lysine and carboxymethylcellulose. Cancer Res. 45:1058; Wiltrout, R. H., Salup, R. R., Twilley, T. A., Talmage, J. E. 1985. mmunomodulatory of natural killer activity by polyribonucleotides. J. Biol. Resp. Mod. 4:512), poly (dG,dC) is mitogenic for B cells (Messina, J. P., Gilkerson, G. S., Pisetsky, D. S. 1993. The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens. Cell. Immunol. 147:148) and induces IFN and NK activity (Tocunaga, T., Yamamoto, S., Namba, K. 1988. A synthetic single-stranded DNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killer activity, and suppresses tumor growth. Jpn. J. Cancer Res. 79:682).
Bacterial DNA has also been reported to have immunostimulatory properties. These properties include the induction of cytokines (interferon gamma (IFN γ), alpha (IFN α), beta (IFN β); tumor necrosis factor alpha (TNF α), interleukin 6 (IL6), 12 (IL 12) and 18 (IL 18), as well as the direct stimulation of B cells (Yamamoto, S. et al. 1988. In vitro augmentation of natural killer cell activity of interferon α/β and γ with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn. J. Cancer Res. (Gann) 79: 866-873; Yamamoto S. et al, 1992. DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol. Immunol. 36: 983-997.; Klinman, D. M., Yi, A-K., Beaucage, S. L., Conover, J. and Krieg, A. M., 1996.
CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12 and interferon γ. Proc. Natl. Acad. Sci. USA 93, 2879-2883. Halpern, M. D., et al. 1996. Bacterial DNA induces murine interferon -γ production by stimulation of interleukin-12 and tumor necrosis factor-α. Cell. Immunol. 167: 72-78. Sparwasser, T. et al, 1997. Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-α-mediated shock. Eur. J. Immunol. 27: 1671-1679; Krieg, A. M. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 345-349).
In contrast, it has been reported that mammalian DNA has no significant immune effects (Pisetsky, D. S. 1996. The immunologic properties of DNA. J. Immunol. 156: 421-423 ; Messina et al. 1991. Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J. Immunol. 147: 1759).
Synthetic DNA has also been reported to be immunostimulatory if contains unmethylated CpG motifs. (Yamamoto, S et al.; 1992. Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity. J. Immunol. 148: 4072-4076; Ballas, Z. K., et al.; 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157: 1840-1845; Hartmann, G., Krieg, A. M. 2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164:944; Hartmann, G., Weeratna, R. D., Ballas, Z. K., Payette, P., Blackwell, S., Suparto, I., Rasmussen, W. L., Waldschmidt, M., Sajuthi, D., Purcell, R. H., Davis, H. L., Krieg, A. M. 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164:1617; Verthelyi, D., Ishii, K. J., Gursel, M., Takeshita, F., Klinman, D. M. 2001. Human peripheric blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol. 166:2372). However, one oligonucleotide containing phosphorothioate bonds that lack CpG motifs has been found to have some immunostimulatory activity on human B cells (Liang, H., Nishioka, Y., Reich, C. F., Pisetsky, D. S., Lipsky, P. E. 1996. Activation of human B cells by phosphorothioate oligonucleotides. J. Clin. Invest. 98:1119). This particular non-CpG oligonucleotide containing phosphorothioate bonds is a poy-T chain, 20 nucleotides long. Also, Vollmer et al (Vollmer J, Janosch A, Laucht M, Ballas Z K, Schetter C, Krieg A M. Highly immunostimulatory CpG-free oligodeoxynucleotides for activation of human leukocytes. Antisense Nucleic Acid Drug Dev. 12:165-175, 2002) reported immunostimulation by phosphorothioate poly T ODNS. These authors pointed out that poly T ODNs are only active as phosphorothioate ODNs and have much lower activity than CpG ODNs.
It has now been discovered that non-CpG oligonucleotides containing the following non-palindromic sequence motif:X1X2X3X4X5X6X7X8,wherein X1 is C,T,G or A (preferably T or C); X2 is C,T,G or A; X7 is C,T,G or A (preferably G); at least three, and preferably all, of X3, X4, X5, X6 and X8 are T; with the proviso that, in the motif, a C does not precede a G, have potent immunostimulatory activity. Therefore, these oligonucleotides can be administered to subjects to treat “immune system deficiencies” or in conjunction with a vaccine, as adjuvants, to boost the immune system in order to have a better response to the vaccine or administered to subjects to increase the responsiveness to tumors.